1. Field of the Invention
The invention relates to synthetic oligonucleotides that have an inhibitory effect upon pathogens. More particularly, the invention relates to anti-pathogen synthetic oligonucleotides having superior inhibitory properties relative to known oligonucleotides.
2. Summary of the Related Art
The use of synthetic oligonucleotides as anti-infecting agents has recently developed into a promising field. Agrawal, Trends in Biotechnology 10:152-158 (1992), reviews the development of antisense oligonucleotides as antiviral agents. Synthetic oligonucleotides now show considerable promise not only as antiviral agents, but as inhibitors of other pathogens as well. Rapaport et al., Proc. Natl. Acad. Sci. USA 89:8577-8580 (1992), discloses antimalarial activities of oligonucleotide phosphorothioates against Plasmodium falciparum.
Due to the great promise of synthetic oligonucleotides as anti-infective agents, considerable interest has developed in improving the pharmacological properties of such compounds. Many such efforts have involved introducing modified internucleoside linkages into oligonucleotides, thereby providing increased resistance to nucleolytic degradation and improving biostability. Agrawal et al., Proc. Natl. Acad. Sci. USA 85:7079:7083 (1988), teaches inhibition of human immunodeficiency virus (HIV) propagation by oligodeoxynucleotide phosphorothioates and phosphoramidites. Sarin et al., Proc. Natl. Acad. Sci. USA 85:7448-7451 (1988), discloses inhibition of HIV by oligodeoxynucleotide methylphosphonates. Padmapriya and Agrawal, Bioorganic and Medicinal Chemistry Letters 3:761-764 (1993), discloses oligonucleotides having novel methylphosphonothioate internucleoside linkages.
Goodchild and Zamecnik (U.S. Pat. No. 4,806,463) disclose antisense oligonucleotides that inhibit HTLV-III replication and protein expression. These oligonucleotides are targeted to highly conserved regions in the HTLV-III genome. Among the sites targeted are: a) the rRNAlys primer binding site, b) regions of the HTLV-III genome vicinal in the 5xe2x80x2 direction to the rRNAlys primer binding site, c) the tRNAlys primer binding site and regions of the HTLV-III genome vicinal in the 5xe2x80x2direction to the tRNAlys primer binding site, d) the mRNA donor splice sites, e) the mRNA acceptor splice sites, f) the initiator codon for the gag gene, g) the initiator condon for the env gene, h) the initiator codon for the tat gene, i) the initiator codon for the sor gene, j) the initiator codon for the 3xe2x80x2 orf gene, k) the cap nucleotide of the HTLV-III genome, l) the art gene or portions thereof, m) and the region of the HTLV-III genome encoding a grameshift.
Other modifications, not necessarily involving solely the use of modified phosphodiester internucleotide linkage, have included the introduction of chemical blocking structures at the 3xe2x80x2 end of oligonucleotides. Temsamani et al., Annals of the New York Academy of Sciences 660:318-320 (1992), teaches that 3xe2x80x2-capped oligonucleotide phosphorothioates have superior in vivo pharmacokinetics and bioability relative to uncapped oligonucleotide phosphorothioates. Tang et al., Nucleic Acids Res. 21:2729 (1993), teaches that oligonucleotides having 3xe2x80x2 terminal hairpin structures demonstrate superior biostability. Koga et al., J. Org. Chem. 56:3757-3759 (1991), reports improved nuclease resistance for alternating xcex1,xcex2-oligothymidylates having alternating (3xe2x80x2 to 3xe2x80x2) and (5xe2x80x2 to 5xe2x80x2) internucleotide phosplhodiester linkages. Seliger et al., Nucleosides and Nucleotides 10:469-477 (1991), and Ortigao et al., Antisense Research and Development 2:129-146 (1992), disclose improved resistance to nucleolytic degradation for oligodeoxynucleotides having single terminal 3xe2x80x2 to 3xe2x80x2 and 5xe2x80x2 to 5xe2x80x2 linkage inversions.
Methods for synthesizing oligonucleotides having at least one 3xe2x80x2 to 3xe2x80x2 internucleoside linkage can involve the use of modified nucleoside monomers that allow 5xe2x80x2 to 3xe2x80x2 synthesis or the use of linkers from which dual 3xe2x80x2 to 5xe2x80x2 synthesis of oligonucleotides having 3xe2x80x2 to 3xe2x80x2 and 5xe2x80x2 to 5xe2x80x2 linkages using commercially available 5xe2x80x2 phosphoramidite nucleoside monomers. CLONETECHniques (April 1993) discloses synthesis of oligonucleotides having a single 3xe2x80x2 to 3xe2x80x2 internucleoside linkage using a commercially available branched linker. Horne and Dervan, J. Am. Chem. Soc. 112:2435-2437 (1990); Luebke and Dervan, Nucleic Acids Res. 20:3005-3009 (1992); and van de Sande et al., Science 241:551-557 (1988), disclose similar synthesis of oligonucleotides having a single 3xe2x80x2 to 3xe2x80x2 linkage for the purpose of studying alternate strand triple helix formation or parallel stranded DNA.
Certain potential problems for effective use of synthetic oligonucleotides as anti-infective therapeutics arise, however, from the nature of the target, rather than the oiligonucleotide, and are thus not addressed by improving the biostability of the oligonucleotide. One such problem is the potential for an infective agent to escape oligonucleotide-mediated therapy by mutation of the target sequence, thereby reducing the ability of the oligonucleotide to interact with the target sequence. For example, Lisziewicz et al., Proc. Natl. Acad. Sci. USA 89:11209-11213 (1992) teaches that a splice acceptor site antisense oligonucleotide initially suppressed HIV in infected MOLT-3 cells, but that after 25 days, viral breakthrough of the suppression was observed. This report suggests that combined or sequential treatment with oligonucleotides complementary to distinct targets may be useful in avoiding viral breakthrough.
There remains a need for additional means of providing biostability to synthetic oligonucleotides that inhibit pathogens. There also remains a need for new ways to avoid mutation-derived breakthrough of pathogens. Ideally, an oligonucleotide should be developed that overcomes both of these problems.
The invention relates to synthetic oligonucleotides that have an inhibitory effect upon pathogens. The invention provides anti-pathogen synthetic oligonucleotides that have superior inhibitory properties, relative to known oligonucleotides. The superior inhibitory properties of oligonucleotides according to the invention are the result of the primary structural feature of such oligonucleotides, which is the linking together of two or more oligonucleotide sequences that are complementary to one or more essential gene of one or more pathogen.
In a first aspect, the invention provides oligonucleotides having two or more identical oligonucleotide sequences linked together, wherein each oligonucleotide sequence is complementary to the same target sequence of a pathogen. The target sequence is the sequence of a portion of a gene or regulatory sequence that is essential for the propagation of the pathogen. According to this aspect of the invention, the identical oligonucleotide sequences may be linked together in a 5xe2x80x2 to 3xe2x80x2 configuration or in a 3xe2x80x2 to 3xe2x80x2 configuration. In this latter configuration, the oligonucleotide is highly resistant to nucleolytic degradation.
In a second aspect, the invention provides oligonucleotides having two or more different oligonucleotide sequences linked together, wherein each oligonucleotide sequence is complementary to a different target sequence of the same pathogen. The different oligonucleotide sequences may be complementary to different portions of the same gene or regulatory sequence or they may be complementary to different genes and/or regulatory sequences. Oligonucleotides according to this aspect of the invention provide improved resistance to mutation-induced pathogen escape from the inhibitory effect of the oligonucleotide. The different oligonucleotide sequences may be linked together in 5xe2x80x2 to 3xe2x80x2 or 3xe2x80x2 to 3xe2x80x2 configurations, with the latter configuration providing greatly increased resistance to nucleolytic degradation.
In a third aspect, the invention provides oligonucleotides having two or more different oligonucleotide sequences linked together, wherein one or more oligonucleotide sequence is complementary to a target sequence from one pathogen and one or more oligonucleotide sequence is complementary to a gene or regulatory sequence from another pathogen. Oligonucleotides according to this aspect of the invention provide combined treatment for infections involving two different pathogens. The different oligonucleotide sequences may be linked together in 5xe2x80x2 to 3xe2x80x2 or 3xe2x80x2 to 3xe2x80x2 configurations, with the latter configuration providing greatly increased resistance to nucleolytic degradation.
In a fourth aspect, the invention provides oligonucleotides having two or more different oligonucleotide sequences linked together, wherein one or more oligonucleotide sequence is complementary to a gene or regulatory sequence from one strain of a pathogen and one or more oligonucleotide sequence is complementary to a gene or regulatory sequence from another strain of the same pathogen. Oligonucleotides according to this aspect of the invention provide the advantage of being effective at inhibiting two or more strains of the pathogen, thus allowing the oligonucleotide to be used as an inhibitory agent even before the particular strain of the pathogen is known.
Those skilled in the art will appreciate that combinations of the various aspects of the invention may be employed within a single oligonucleotide to provide an oligonucleotide having superior characteristics for the treatment of particular disease conditions. They will also recognize that if the identical or different oligonucleotide sequences of the first three aspects of the invention have a 3xe2x80x2 terminal ribonucleotide, then the identical or different oligonucleotides can be linked by 5xe2x80x2 to 3xe2x80x2, 5xe2x80x2 to 2xe2x80x2, 2xe2x80x2 to 3xe2x80x2, 3xe2x80x2 to 2xe2x80x2, or 3xe2x80x2 to 3xe2x80x2 linkages.
It is an object of the invention to provide anti-pathogen oligonucleotides that reduce or eliminate mutation-induced pathogen escape from the inhibitory effect of the oligonucleotide. It is a further object of the invention to provide anti-pathogen oligonucleotides that can inhibit more than one pathogen simultaneously, particularly where such pathogens commonly coinfect host organisms in nature.
The foregoing merely summarizes certain aspects of the present invention and is not intended, nor should it be construed, to limit the invention in any way.
All patents and publications cited herein are hereby incorporated by reference in their entirety.